Biosensors designed to analyze a sample by an electrochemical or optical method are widely used. An example of biosensors designed to analyze a sample by an electrochemical method (see Patent Document 1, for example) is a biosensor 9 shown in FIG. 12 of the present application.
The illustrated biosensor 9 includes a substrate 92 formed with a working electrode 90 and a counter electrode 91, and a cover 94 bonded to the substrate via a spacer 93. The biosensor 9 further includes a flow path 95 defined by the substrate 92, the spacer 93 and the cover 94. The flow path 95 is used for moving a sample by capillary force and formed with a reagent portion 96.
The reagent portion 96 connects the ends of the working electrode 90 and the counter electrode 91 and contains oxidoreductase. The oxidoreductase catalyzes the reaction of taking electrons from glucose, for example. The electrons taken from the glucose are supplied to the working electrode 90. The amount of electrons supplied to the working electrode 90 is measured as the responsive current by utilizing the working electrode 90 and the counter electrode 91.
The four methods described below are typical methods for forming a reagent portion 96, i.e., the methods for immobilizing oxidoreductase (see Non-patent document 1, for example).
In the first method, a material liquid containing oxidoreductase is applied to an intended portion of a target, and then the material liquid is dried. In this way, the oxidoreductase is immobilized to the intended portion of the target.
In the second method, oxidoreductase is immobilized to an intended portion of a target by using a cross-linker such as glutaraldehyde.
In a third method, oxidoreductase is contained in a polymer such carboxymethylcellulose (CMC), and then the oxidoreductase immobilized together with the polymer.
In a fourth method, oxidoreductase is dispersed in a conductive material such as a carbon paste, and the resultant paste is applied to an intended portion of a target, to immobilize the oxidoreductase.
However, with the conventional oxidoreductase-immobilizing methods described above, oxidoreductase fails to be immobilized in a manner such that the active sites are oriented (located) to exhibit efficient activity of the oxidoreductase. In other words, the conventional methods have a drawback that the immobilization is not performed with the orientation of the oxidoreductase being controlled. Specifically, with the conventional methods, active sites of oxidoreductase existing adjacent to each other may face each other or proteins may aggregate each other so that the active site exists within the aggregate. As a result, the ratio of the oxidoreductase (active site) which can be utilized efficiently is relatively low. Accordingly, the probability that oxidoreductase comes into contact with a substrate is relatively low, so that the activity of the immobilized oxidoreductase as a whole is low. Thus, to exhibit the intended function of the immobilized oxidoreductase, the amount of oxidoreductase to be loaded needs to be increased, which is disadvantageous in terms of cost. Particularly, since oxidoreductases are generally expensive, the increase in the amount of oxidoreductase to be loaded leads to a considerably disadvantageous cost increase.
Moreover, since the orientation of oxidoreductase cannot be controlled, the ratio of the actually usable oxidoreductase varies among biosensors even when the same amount of oxidoreductase is loaded. As a result, when the conventional immobilization methods which cannot control orientation are employed, the measurement results vary among biosensors.
Further, in the above-described biosensor 9, electrons taken from the substrate at the active site of the oxidoreductase are transferred to the working electrode 90. However, when the orientation of the oxidoreductase is random, the efficiency of electron transfer from the oxidoreductase to the working electrode is poor. Thus, when the immobilization methods by which the orientation of the oxidoreductase becomes random are employed, an electron mediator needs to be added to mediate the electron transfer between the oxidoreductase and the working electrode 90. Therefore, the biosensor 9 provided by immobilizing oxidoreductase by a conventional method is disadvantageous in terms of cost, because it requires an electron mediator. Further, as the electron mediator, metal complexes such as potassium ferrocyanide are used some of which have an adverse effect on the human body. Thus, it is not desirable to use an electron mediator for such an analytical tool as the biosensor 9.
Patent document 1: JP-B-H08-10208
Non-patent document 1: MIZUTANI Fumio, “Application of enzyme-modified electrodes to biosensors,” BUNSEKI KAGAKU, Vol. 48, No. 9 pp. 809-821, The Japan Society for Analytical Chemistry, September, 1999.